Development and Evaluation of Self-Microemulsifying Drug Delivery System for Improving Oral Absorption of Poorly Water-Soluble Olaparib

The purpose of this study is to develop and evaluate a self-microemulsifying drug delivery system (SMEDDS) to improve the oral absorption of poorly water-soluble olaparib. Through the solubility test of olaparib in various oils, surfactants and co-surfactants, pharmaceutical excipients were selected. Self-emulsifying regions were identified by mixing the selected materials at various ratios, and a pseudoternary phase diagram was constructed by synthesizing these results. The various physicochemical properties of microemulsion incorporating olaparib were confirmed by investigating the morphology, particle size, zeta potential, drug content and stability. In addition, the improved dissolution and absorption of olaparib were also confirmed through a dissolution test and a pharmacokinetic study. An optimal microemulsion was generated in the formulation of Capmul® MCM 10%, Labrasol® 80% and PEG 400 10%. The fabricated microemulsions were well-dispersed in aqueous solutions, and it was also confirmed that they were maintained well without any problems of physical or chemical stability. The dissolution profiles of olaparib were significantly improved compared to the value of powder. Associated with the high dissolutions of olaparib, the pharmacokinetic parameters were also greatly improved. Taken together with the results mentioned above, the microemulsion could be an effective tool as a formulation for olaparib and other similar drugs.


Introduction
The Biopharmaceutics Classification System (BCS) classifies drugs into four groups based on their solubility and gastro-intestinal membrane permeability in an aqueous

Materials and Methods
To explain the research design, a concise overview is shown in Scheme 1.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 3 of 14 in vivo pharmacokinetic profile. In addition, the optimal SMEDDS formulation was compared with Lynparza ® (a commercialized product), because our research is the first application of a microemulsion system to OLA.

Materials and Methods
To explain the research design, a concise overview is shown in Scheme 1.

Scheme 1.
Overview of the research design in this study.

Materials
OLA was supplied by ScinoPharm Taiwan

Solubility Test
The solubilities of OLA were quantitatively analyzed in various aqueous solutions (distilled water, pH 1.2 and pH 6.8 solution), oils (Capmul ® MCM EP/NF, cotton seed oil, mineral oil, Labrafil ® M 1944 CS, Labrafac ® PG and oleic acid) and surfactants (Kolliphor ® EL, Tween 80, Span 80, Labrasol ® , Transcutol ® HP, Plurol Oleique ® CC 497, isopropyl myristate and PEG 400). An excess amount of OLA was poured into each microtube containing 1.0 mL of aqueous solution, oil or surfactant. Using a Daihan RT-10 rotary mixer (Seoul, Republic of Korea), each of the blends of materials were shaken for 72 h at room temperature. Then, the tubes were centrifuged at 10,000× g rpm for 15 min using a Daihan CF-10 microcentrifuge (Seoul, Republic of Korea) to separate the undissolved OLA. The obtained supernatants were filtered using a 0.45 µm nylon membrane and quantitatively evaluated by a high-performance liquid chromatography system. A quantitative analysis of OLA in each filtered solution was conducted using a Hitachi Chromaster ® HPLC system (Tokyo, Japan) equipped with a UV detector and LB-Science Supersil 120 ODS II C 18 column (4.6 × 250 mm, 5 µm; Dalian, China). The mobile phase comprised a mixture solution (acetonitrile: distilled water, 50:50 (v/v)). The flow rate of the mobile phase solution was 1.0 mL/min, and the column temperature was maintained at 30 • C. The detection UV wavelength was set at 200 nm.

Self-Emulsification and Pseudoternary Phase Diagram
Considering the solubility results for OLA in the solubility test, Capmul ® MCM EP/NF, Labrasol ® and PEG 400 were selected as the oil, surfactant and co-surfactant, respectively. To identify the self-emulsifying region of SMEDDS, the self-emulsification of a variety of compositions was meticulously observed. We added 50 µL of a mixture of Capmul ® MCM EP/NF, Labrasol ® and PEG 400 to 50 mL of distilled water, gently mixed them using a magnetic stirrer and observed their self-emulsification with naked eyes. If we obtained a nearly transparent homogenous emulsion without phase separation, it was judged that the microemulsion was successful. On the contrary, if we observed a turbid mixture or no emulsification progress, it was judged that the microemulsion was a failure. Based on the obtained self-emulsification observations, a pseudoternary phase diagram was constructed.

Preparation of SMEDDS
The SMEDDS formulations were selected based on the identification of a self-emulsifying region in the pseudoternary phase diagram. The formulations were prepared by mixing a certain amount of OLA, oil, surfactant and co-surfactant. A total of 100 mg of OLA was dissolved in a 3 mL mixture solution of Capmul ® MCM EP/NF, Labrasol ® and PEG 400, and mildly stirred until the mixture was clear. The obtained solutions were stored at room temperature until their physico-chemical characterizations.

Morphological Analysis
The morphologies of the fine emulsion droplets of the prepared SMEDDS formulation were observed using a Hitachi H-7500 transmission electron microscope (TEM) system (Tokyo, Japan). After the dilution of the sample solution with distilled water, the samples were deposited on the carbon-coated grid and dried at room temperature. Then, the dried samples were stained with 2% (w/v) phosphotungstic acid solution for 2 min prior to observation.

Droplet Size and Zeta Potential Analysis
The droplet size distribution and zeta potential of the prepared microemulsions were evaluated by an Otsuka ELSZ-100 particle size analyzer (Tokyo, Japan). A total of 5 µL of the prepared microemulsion was added to 50 mL of distilled water and the diluted sample was analyzed at room temperature (n = 3). The particle size values and distribution were calculated from the intensity-weighted distribution and polydispersity index (PDI), respectively.

Stability Test
To evaluate the storage stability of the SMEDDS formulation, the samples were tightly sealed in 5 mL glass vials. According to the ICH guidelines (Q1), the samples were stored for 3 months under long-term storage conditions (25 ± 2 • C, 60 ± 5% RH) and accelerated storage conditions (40 ± 2 • C, 75 ± 5% RH). A storage stability test was conducted on the physical application, emulsification, droplet distribution, zeta potential and OLA contents at 1 and 3 months after the initial time. Each sample was evaluated three times and the data were processed.

In Vitro Dissolution Test
An in vitro dissolution study of OLA was conducted using a Kukje KDT-600 dissolution tester (Goyang, Republic of Korea) under the USP apparatus II (paddle method). The dissolution media were HCl buffer (pH 1.2), phosphate buffer (pH 6.8) and distilled water. The paddles were rotated at 100 rpm and the temperature was 37 • C. The SMEDDS system (equivalent to OLA 10 mg) was filled into capsules (hard gelatin) prior to the dissolution test and compared with the OLA powder. During the dissolution test, each dissolution medium (3 mL) was taken at predetermined times (5,10,15,30,45,60, 90 and 120 min) and filtered through a 0.45 µm syringe filter. The obtained aliquots were quantitatively analyzed using the HPLC system as described in Section 2.2. After taking dissolution samples, an equal volume of the dissolution medium was replaced at each time point and all the experiments were conducted three times. The statistical significances between the results of the OLA powder and SMEDDS formulation were confirmed by the student's t-test method using Jandel scientific Sigmaplot version 12.0 (San Rafael, CA, USA).

In Vivo Pharmacokinetic Study
To confirm the improvement of the oral absorption of OLA contained in the SMEDDS formulation, a rat pharmacokinetic study of the OLA powder, SMEDDS formulation and Lynparza ® tablet was conducted. The rats used in this experiment were fasted overnight, but water was freely consumed. Blood samples were taken from the jugular vein using a 1.0 mL syringe treated with heparin at predetermined times (0, 30, 60, 120, 240, 480 and 1440 min) after the oral administration of each formulation. The blood samples were centrifuged at 7000× g rpm and 4 • C for 10 min, and the obtained plasma was stored at −80 • C until the quantitative analysis.
A total of 50 µL of the plasma samples was added to 150 µL of an internal standard solution (carbamazepine 200 ng/mL in acetonitrile), then mixed on a vortex mixer for 3 min. After centrifugation for 10 min at 13,000× g rpm at 4 • C, the supernatants were transferred into new LC vials and analyzed by injecting 5 µL of the supernatants into an LC-MS/MS system. The autosampler was set at 10 • C during the analysis. The chromatographic separation of OLA was conducted on a Gemini-NX C18 column (50 × 2.0 mm, 3 µm particle size, 110 Å; Phenomenex Inc., Torrance, CA, USA) using an isocratic elution condition of acetonitrile and 10 mM of ammonium formate buffer (80:20, v/v) at a flow rate of 0.3 mL/min and a column oven temperature of 40 • C, using the Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). The method of mass spectrometry known as multiple reaction monitoring (MRM) was utilized for the analytical procedure, and the following parameters were used: an ion-spray voltage of + 5500 V at 550 • C; a decluttering potential of 111 V; an entrance potential of 11 V; a collision energy of 37 V and a mass transition m/z of 435.1 > 281.3 for OLA.
The following pharmacokinetic parameters were calculated using the WinNonlin program version 5.0 (Princeton, NJ, USA): the time to reach the maximum plasma concentration (T max ); the maximum plasma concentration (C max ); the area under the plasma concentration-time curve to the last time point or infinity time (AUC last or AUC inf ,) and the half-life (T 1/2 ). Additionally, the relative oral bioavailability (BA) was calculated by dividing the AUC for the SMEDDS formulation or the Lynparza ® tablet by that of OLA. All data were expressed as means ± S.D. The student's t-test was conducted to determine statistically significant differences (p < 0.05) among the tested groups.

In Vitro Cytotoxicity Assay
The cell viability values of the formulated SMEDDS system were evaluated by an MTT assay using the CT26 cells (human colon carcinoma). RPMI 1640 media supplemented with FBS (10%), penicillin (100 units/mL) and streptomycin (100 mg/mL) were used as the culture media for the CT26 cell line at 37 • C and 5% CO 2 . The cultured cell lines were placed at a density of about 1 × 10 4 cells/well in well microplates and allowed to adhere onto the bottom of the wells. The cell lines attached to the wells were washed with phosphate buffered saline (PBS) and incubated for 24 h with various concentrations of SMEDDS solution without OLA. The treated cells were washed with PBS, treated with the MTT solution (5 mg/mL in PBS) and incubated again at 37 • C for 6 h. The cell viability (%) in the obtained solution was evaluated at 570 nm using a UV spectrophotometer and compared with untreated control samples.

Solubility Test of OLA
Solubility tests of OLA in various aqueous solutions, oils and surfactants were performed ( Table 1). The aqueous solubility of OLA was 0.073 ± 0.25 mg/mL in distilled water, 0.063 ± 0.57 mg/mL at a pH of 1.2 and 0.059 ± 0.42 mg/mL at a pH of 6.8, consistently very low regardless of pH. These results show that, even after the oral administration of OLA with a daily dose of 800 mg, most of the OLA is not dissolved in the GI tract and is precipitated in an insoluble state, which may lead to very low oral absorption. In general, it is known that the lower the solubility of a drug, the lower the bioavailability, which increases the administered dose, as well as side effects [30]. Improving the drug solubility using an appropriate solubilizer may increase the oral absorption, thereby decreasing the administrated dose. As a result, this study conducted a screening test of excipients frequently used in the pharmaceutical field to search for substances that can improve the solubility of OLA. Among various oils, the solubility of Capmul ® MCM EP/NF was about 574 times that of distilled water (41.68 ± 0.02 mg/mL vs. 0.07 ± 0.25 mg/mL). In the case of surfactants, the solubilities of PEG 400 and Labrasol ® were 47.66 ± 0.38 mg/mL and 36.60 ± 0.07 mg/mL, respectively, which were improvements of about 657.3 times and 504.8 times, respectively, compared to distilled water. Through the excipient solubility screening test described above, Capmul ® MCM EP/NF, PEG 400 and Labrasol ® were selected as the oil, surfactant and co-surfactant to be used in the production of OLA SMEDDS.

Construction of a Pseudoternary Phase Diagram
A self-emulsification assessment was performed using the selected oil (Capmul ® MCM EP/NF) and surfactant (PEG 400 or Labrasol ® ). Various SMEDDS mixtures were prepared by diversifying the ratio of the selected substances to proceed with various evaluations. By constructing a pseudoternary phase diagram based on the obtained results, self-emulsifying regions could be identified. The region shown on Figure 1 represents a stable self-emulsifying region, either clear or slightly bluish. When the oil ratio was 5-30%, an excellent microemulsion was observed.
It was confirmed that the higher the oil ratio, the larger the droplet size of the emulsion. When checking the pseudoternary phase diagram, the higher the ratio of Labrasol ® or PEG 400 that was used as surfactant, rather than Capmul ® MCM EP/NF, the better the emulsions that were generated. This phenomenon is mentioned in several studies, revealing that as the proportion of surfactant and co-surfactant increases, the interface of the emulsion becomes more stabilized and condensed [35]. Based on the pseudoternary phase diagram, Capmul ® MCM EP/NF 10%, Labrasol ® 80% and PEG 400 10% were selected as the optimal SMEDDS production ratio for OLA. Considering the solubility of OLA in each substance, a SMEDDS formulation containing 100 mg of OLA in a mixture of Capmul ® MCM EP/NF 0.3 mL, Labrasol ® 2.4 mL and PEG 400 0.3 mL was obtained. Using this resultant formulation, several characterizations were investigated.
MCM EP/NF) and surfactant (PEG 400 or Labrasol ® ). Various SMEDDS mixtures were prepared by diversifying the ratio of the selected substances to proceed with various evaluations. By constructing a pseudoternary phase diagram based on the obtained results, self-emulsifying regions could be identified. The region shown on Figure 1 represents a stable self-emulsifying region, either clear or slightly bluish. When the oil ratio was 5-30%, an excellent microemulsion was observed.
It was confirmed that the higher the oil ratio, the larger the droplet size of the emulsion. When checking the pseudoternary phase diagram, the higher the ratio of Labrasol ® or PEG 400 that was used as surfactant, rather than Capmul ® MCM EP/NF, the better the emulsions that were generated. This phenomenon is mentioned in several studies, revealing that as the proportion of surfactant and co-surfactant increases, the interface of the emulsion becomes more stabilized and condensed [35]. Based on the pseudoternary phase diagram, Capmul ® MCM EP/NF 10%, Labrasol ® 80% and PEG 400 10% were selected as the optimal SMEDDS production ratio for OLA. Considering the solubility of OLA in each substance, a SMEDDS formulation containing 100 mg of OLA in a mixture of Capmul ® MCM EP/NF 0.3 mL, Labrasol ® 2.4 mL and PEG 400 0.3 mL was obtained. Using this resultant formulation, several characterizations were investigated.

Characterization of SMEDDS
The morphology of the prepared SMEDDS was observed using TEM. Figure 2 shows that the microemulsion droplets are well dispersed. The mean droplet size was 141.1 ± 0.2 nm, and the polydispersity index was very small, 0.19. In general, having a PDI value of 0.2 or less in a dispersed system means that the particle size distribution is quite narrow [36]. The zeta potential was measured at −15.8 ± 1.1 mV, and it has been reported that nano-sized dispersed systems with a zeta potential lower than 10 mV can maintain physical stability and improve drug efficacy by improving in vivo absorption [37]. Judging by the obtained test results and the results of various other studies, it was expected that the manufactured OLA SMEDDS system could show high physical stability and oral absorption of OLA because it had the appropriate physical properties.

Characterization of SMEDDS
The morphology of the prepared SMEDDS was observed using TEM. Figure 2 shows that the microemulsion droplets are well dispersed. The mean droplet size was 141.1 ± 0.2 nm, and the polydispersity index was very small, 0.19. In general, having a PDI value of 0.2 or less in a dispersed system means that the particle size distribution is quite narrow [36]. The zeta potential was measured at −15.8 ± 1.1 mV, and it has been reported that nano-sized dispersed systems with a zeta potential lower than 10 mV can maintain physical stability and improve drug efficacy by improving in vivo absorption [37]. Judging by the obtained test results and the results of various other studies, it was expected that the manufactured OLA SMEDDS system could show high physical stability and oral absorption of OLA because it had the appropriate physical properties.

Stability Study of SMEDDS
To evaluate the stability of the obtained SMEDDS formulation, the physico-chemical properties were checked while storing the fabricated samples under the long-term stability test conditions (25 ± 2 °C, 60 ± 5% RH) and accelerated stability test conditions (40 ± 2

Stability Study of SMEDDS
To evaluate the stability of the obtained SMEDDS formulation, the physico-chemical properties were checked while storing the fabricated samples under the long-term stability test conditions (25 ± 2 • C, 60 ± 5% RH) and accelerated stability test conditions (40 ± 2 • C, 75 ± 5% RH) ( Table 2). During the storage period, the formulation's physical stability was confirmed as it maintained its clear liquid state without drug precipitation and phase separation. In addition, when the SMEDDS formulation was dispersed in distilled water, the self-emulsification was also confirmed to have maintained its initial state. For 3 months, the droplet size increased from 141. 1 ± 0.2 nm to 166.1 ± 2.7 nm, and the zeta potential slightly increased from −13.5 ± 0.8 mV to −18.8 ± 0.4 mV, but the nano-sized dispersion still remained stable. Lipid-or oil-based particulate systems can maintain their physical stability thanks to the electrostatic effects of the surface charge and steric stabilization from the PEG chain [38]. The physical stability of the microemulsions manufactured in this study could be maintained by a negative charge with an absolute value of more than 10 mV and steric stabilization generated by the addition of PEG. The drug contents were 101.0 ± 0.2% and 98.5 ± 0.2% in long term storage and accelerated storage, respectively, which is not significantly different from the initial value (101.1 ± 0.5%). Because the SMEDDS system does not contain water in the pharmaceutical formulation, it is generally known to have a relatively high stability compared to other liquid-type dosage formulations [23]. Taken together, these results show that the self-emulsification behavior was maintained and the physical properties of the SMEDDS formulation did not change significantly.

In Vitro Dissolution Study
The in vitro dissolution profiles of OLA from the SMEDDS formulation were monitored and compared with the OLA powder under different pH conditions (pH 1.2, pH 6.8 and distilled water) for 2 h (Figure 3). In the pH 1.2 solution, the dissolutions of the OLA powder were only 6.09 ± 0.27% within 5 min and 45.39 ± 3.97% at 120 min. On the other hand, that of OLA in the SMEDDS formulation was 64.38 ± 0.48% within 5 min, showing a very rapid dissolution profile, followed by a final dissolution of 74.81 ± 1.87% at 120 min. Similar results to those at a pH of 1.2 were also obtained at a pH of 6.8 and in distilled water. At a pH of 6.8 and in distilled water, the dissolution values of OLA in the SMEDDS system were 84.20 ± 0.31% and 67.93 ± 0.64%, respectively, for the initial 5 minutes. In contrast, those of the OLA powders were very low at 5.70 ± 0.59% and 4.58 ± 0.14%, respectively. According to the solubility results, the solubility of OLA is independent of the pH conditions. Therefore, it is estimated that the dissolution profiles at a pH of 1.2, a pH of 6.8 and in distilled water are all similar. The dissolutions of OLA contained in SMEDDS at the initial 5 min had improved 10.57 times (pH 1.2), 14.78 times (pH 6.8) and 14.83 times (distilled water) compared to the OLA powder, and the final dissolution rates (at 120 min) had improved 1.65 times (pH 1.2), 1.57 times (pH 6.8) and 1.52 times (distilled water). The improved dissolution rates of the drugs contained in SMEDDS formulations has been reported in several documents. These results showed that the improved dissolutions of the SMEDDS formulation containing OLA were because of the increased specific surface area of nano-sized microemulsion droplets and the solubilization effects of optimal oils and Pharmaceutics 2023, 15, 1669 9 of 13 surfactants. As such, the SMEDDS formulation showed an improved dissolution profile compared to the OLA powder, which would lead to the enhanced oral adsorption of OLA.
at the initial 5 min had improved 10.57 times (pH 1.2), 14.78 times (pH 6.8) and 14.83 times (distilled water) compared to the OLA powder, and the final dissolution rates (at 120 min) had improved 1.65 times (pH 1.2), 1.57 times (pH 6.8) and 1.52 times (distilled water). The improved dissolution rates of the drugs contained in SMEDDS formulations has been reported in several documents. These results showed that the improved dissolutions of the SMEDDS formulation containing OLA were because of the increased specific surface area of nano-sized microemulsion droplets and the solubilization effects of optimal oils and surfactants. As such, the SMEDDS formulation showed an improved dissolution profile compared to the OLA powder, which would lead to the enhanced oral adsorption of OLA.

In Vivo Pharmacokinetic Study and In Vitro Cytotoxicity
The in vivo pharmacokinetic behavior of OLA with SMEDDS, a marketed product (Lynparza ® tablet) and OLA powder was investigated in rats. The mean plasma concentration profiles of OLA were plotted as a function of time, as presented in Figure 4. The mean pharmacokinetic parameters of OLA absorption are summarized in Table 3. The Cmax and AUClast of OLA powder were 83.1 ± 49.6 ng/mL and 384.1 ± 136.2 ng·h/mL, respectively. The Cmax and AUClast of the Lynparza ® tablet were 335.7 ± 100.2 ng/mL and 518.2 ± 154.9 ng·h/mL, respectively. On the other hand, the Cmax and AUClast following SMEDDS administration were 351.2 ± 173.3 ng/mL and 1242.7 ± 420.3 ng·h/mL, a significant increase of 4.2 and 3.2, respectively, compared to the OLA powder. When compared with the Lynparza ® tablet, the Cmax was similar, but the AUClast was significantly increased, by 2.4. The AUCinf of the OLA powder and the Lynparza ® tablet were 634.0 ± 437.2

In Vivo Pharmacokinetic Study and In Vitro Cytotoxicity
The in vivo pharmacokinetic behavior of OLA with SMEDDS, a marketed product (Lynparza ® tablet) and OLA powder was investigated in rats. The mean plasma concentration profiles of OLA were plotted as a function of time, as presented in Figure 4. The mean pharmacokinetic parameters of OLA absorption are summarized in Table 3. The C max and AUC last of OLA powder were 83.1 ± 49.6 ng/mL and 384.1 ± 136.2 ng·h/mL, respectively. The C max and AUC last of the Lynparza ® tablet were 335.7 ± 100.2 ng/mL and 518.2 ± 154.9 ng·h/mL, respectively. On the other hand, the C max and AUC last fol-lowing SMEDDS administration were 351.2 ± 173.3 ng/mL and 1242.7 ± 420.3 ng·h/mL, a significant increase of 4.2 and 3.2, respectively, compared to the OLA powder. When compared with the Lynparza ® tablet, the C max was similar, but the AUC last was significantly increased, by 2.4. The AUC inf of the OLA powder and the Lynparza ® tablet were 634.0 ± 437.2 ng·h/mL and 522.0 ± 155.9 ng·h/mL, respectively. The AUC inf of SMEDDS was 1427.3 ± 460.3 ng·h/mL, which was an increase of 2.25 compared to the OLA powder (634.0 ± 437.2 ng·h/mL) and of 2.73 compared to the Lynparza ® tablet (522.0 ± 155.9 ng·h/mL), respectively. The T max of SMEDDS was slightly delayed compared to the Lynparza ® tablet, but the T 1/2 showed there was no significant difference among the formulations. To improve the low oral bioavailability of OLA, several nano-sized formulations (lipo-sphere nanoparticles (LP-NP) by melt dispersion and nano-suspensions (NSP) by solvent evaporation (SE) and wet milling (WM) techniques) have been studied. These dispersion systems are known to be very effective options for improving the oral absorption of poorly water-soluble drugs. The oral AUC of LP-NP, NSP-SE and NSP-WM in rats significantly increased by 1.5, 1.9 and 1.4, respectively [30]. In this study, the AUC of the OLA SMEDDS increased by 3.2, proving that SMEDDS can be used as an effective formulation for improving the oral absorption of OLA (Table 3). In most cases, the toxicity of anticancer drugs increases in proportion to the administered dose [39]. In particular, it is known that the high-dose administration of OLA can cause side effects such as gastrointestinal toxicities, fatigue and anemia, and therefore, dose reduction through formulation improvement can reduce toxicity [40]. As a result, when analyzing the results of the pharmacokinetic study conducted in this study comprehensively, the SMEDDS formulation is expected to significantly reduce the dosage and toxicity compared to the marketed formulation.
Additionally, Capmul ® MCM EP/NF (oil) [41,42], Labrasol ® (surfactant) [43,44] and PEG 400 (co-surfactant) [45,46] are widely employed as biocompatible additives and were incorporated into the pharmaceutical formulations. The use of biocompatible materials in drug development is important because it could reduce the risk of acute toxicity and chronic toxicity. In this study, the SMEDDS formulation containing the above-mentioned safe substances showed almost no cytotoxicity in a safety test using the MTT assay. We confirmed that the cell viability (%) of the pretreated media with a fabricated formulation was over 97% (<3% cell death at all concentrations). This result indicates that the prepared microemulsion has almost no cytotoxic effects on the CT26 cell line. Furthermore, no acute toxic-induced death was observed up to or over 24 h after oral administration in rats.

Conclusions
The optimized SMEDDS containing OLA was developed using Capmul ® MCM EP/NF, Labrasol ® and PEG 400. This formulation system formed a stable microemulsion system for at least 3 months under long-term storage conditions (25 ± 2 °C, 60 ± 5% RH) and accelerated storage conditions (40 ± 2 °C, 75 ± 5% RH). In addition, it showed higher dissolution patterns than OLA powder and even the commercial formulation. The in vivo pharmacokinetic results also showed the improvement of OLA oral absorption. In conclusion, this system suggests the potential use of SMEDDS for the oral administration of OLA or other hydrophobic drugs.  Table 3. Pharmacokinetic parameters of SMEDDS and OLA at an equivalent dose of 10 mg/kg in rats (mean ± S.D.; n = 5); * p < 0.05 and ** p < 0.01 compared with OLA; # p < 0.05 compared with Lynparza ® tablet. In most cases, the toxicity of anticancer drugs increases in proportion to the administered dose [39]. In particular, it is known that the high-dose administration of OLA can cause side effects such as gastrointestinal toxicities, fatigue and anemia, and therefore, dose reduction through formulation improvement can reduce toxicity [40]. As a result, when analyzing the results of the pharmacokinetic study conducted in this study comprehen-sively, the SMEDDS formulation is expected to significantly reduce the dosage and toxicity compared to the marketed formulation.

PK Parameters
Additionally, Capmul ® MCM EP/NF (oil) [41,42], Labrasol ® (surfactant) [43,44] and PEG 400 (co-surfactant) [45,46] are widely employed as biocompatible additives and were incorporated into the pharmaceutical formulations. The use of biocompatible materials in drug development is important because it could reduce the risk of acute toxicity and chronic toxicity. In this study, the SMEDDS formulation containing the above-mentioned safe substances showed almost no cytotoxicity in a safety test using the MTT assay. We confirmed that the cell viability (%) of the pretreated media with a fabricated formulation was over 97% (<3% cell death at all concentrations). This result indicates that the prepared microemulsion has almost no cytotoxic effects on the CT26 cell line. Furthermore, no acute toxic-induced death was observed up to or over 24 h after oral administration in rats.

Conclusions
The optimized SMEDDS containing OLA was developed using Capmul ® MCM EP/NF, Labrasol ® and PEG 400. This formulation system formed a stable microemulsion system for at least 3 months under long-term storage conditions (25 ± 2 • C, 60 ± 5% RH) and accelerated storage conditions (40 ± 2 • C, 75 ± 5% RH). In addition, it showed higher dissolution patterns than OLA powder and even the commercial formulation. The in vivo pharmacokinetic results also showed the improvement of OLA oral absorption. In conclusion, this system suggests the potential use of SMEDDS for the oral administration of OLA or other hydrophobic drugs.  Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (approval code: KW-220914-1).